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Some New Bacterial Diseases of 
Legumes 

and 

The Relationship of the Organisms 
Causing the Same 



THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF 
THE UNIVERSITY OF PENNSYLVANIA IN PARTIAL FUL- 
FILMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



BY 

THOMAS F. MANNS 



PHILADELPHIA 

1914 



Some New Bacterial Diseases of 
Legumes 

and 

The Relationship of the Organisms 
Causing the Same 



THESIS 



PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF 
THE UNIVERSITY OF PENNSYLVANIA IN PARTIAL FUL- 
FILMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



BY 

THOMAS F. MANNS 



PHILADELPHIA 

1914 



m 30 , 9IS 



V 



BULLETIN NO. 108 APRIL, 1915 



"JO 

Delaware College 



Agricultural 



Experiment Station 



Some New Bacterial Diseases of Legumes and 

the Relationship of the Organisms 

Causing the Same 



BY THOMAS F. MANNS 



Newark, Delaware 



Some New Bacterial Diseases of Legumes and 
The Relationship of the Organisms 
Causing the Same* 



By THOMAS F. MANNS 



INTRODUCTION 

The history of phytopathology in relation to bacterial diseases is 
of comparatively recent years. We are indebted to Burnl^ (1877 & 
1878) for the first contribution giving definite experimental evi- 
dence that bacteria may and do cause injuries to plants of a patho- 
logical nature. His work was done upon the twig blight of pear (1878- 
1883). Following closely upon the work of this investigator we find 
others demonstrating what some considered an impossibility, viz. that 
plants could be subject to bacterial diseases (See p. 27, Lehrbuch, Har- 
tig. 1882) 2 . Among Burrill's contemporaries who were at work on 
bacteria that are active pathogens of plants may be mentioned Pril- 
Jieux 3 (his first publication in 1879), Comes 4 (1880), Sorauer 5 (1886) 
Savastano, (1887), Arthur, (1886), Beyerinck 8 (1888), and Wakker 9 
(1883). Erwin F. Smith 10 1899-1901, has been active in removing 
the doubt existing on the possibility of bacteria being active producers 
of disease in plants. His painstaking and exact work on bacteria as 
causes of plant disease will stand as a monumental contribution to the 
science of plant pathology. Within the last decade the number of 
exact workers in the bacterial pathology of plants has increased rap- 
idly ; it is beyond the scope of this paper to mention their names. It 
is sufficient to state that we must admit that certain diseases which 
were at different times assigned to higher fungi as casual organisms 
are now known to be caused by bacteria. 

This bulletin deals with a disease of the sweet pea (Lathyrus odor- 
atus) which has variously been stated to be due to different parasitic 
fungi. It describes also similar diseases upon the clovers (Trifolium 
pp.), culinary beans (Phaseolus spp.), and soy bean (Soja spp.), 
which are here proved to be bacterial in nature. 

^Submitted for publication June, 1913. Presented before the graduate school 
of the University of Pennsylvania as a thesis in partial fulfillment of the re- 
quirements for the degree of Doctor of Philosophy, June, 1913. 



DEVELOPMENT OF PROBLEM 

Mr. J. J. Taubenhaus n having taken up in 1911 the project of 
"Some Fungous Diseases of the Sweet Pea" for thesis work at the 
University of Pennsylvania, met a disease of the sweet pea known 
generally in England and less in the United States as ' ' streak. ' ' Spec- 
imens of diseased plants from Boston, Mass., and from England were 
referred to the writer. The appearance of the disease suggested to him 
that it was of bacterial origin. Preliminary isolation work for the 
parasite readily revealed an abundance of bacteria in the beginning 
lesions. Attempts at infection of sweet peas with pure cultures gave 
positive results in ten days. 

The symptoms of this "streak" disease of the sweet pea were so 
similar to those of a disease we had previously observed on red clover 
that it occurred to us that the latter was possibly of bacterial origin 
and related to the former. A search in the fields in the vicinity of 
Newark, Del., revealed the presence of the formerly observed disease, 
in red, alcike, and mammoth clovers. The "streak" disease was also 
prevalent on several species of Lathyrus, the latter of forage types 
and upon the numerous varieties of sweet peas with which Mr. Tauben- 
haus was working. Platings of beginning lesions from the clovers, the 
different forage species of Lathyrus, and the different varieties of 
sweet peas indicated the causative organism to be a bacterium. 

HISTORY OF "STREAK" 

The disease was first observed, according to a letter in 1912 from 
Mr. T. A. Weston of Orpington, England, by H. J. R. Digges of Dub- 
lin in 1901 or 1905. Mr. Weston states that, in the fall of 1906, he 
had a note of the disease in "The Gardener" describing it as "a new 
disease under the title of 'streak'." In 1908 George Massee 12 in a 
letter to a correspondent who had sent in diseased specimens replied, 
"the disease is of a physiological nature." 

As far as we have been able to obtain literature, it appears that 
ouly since the summer of 1908 has any definite work been done upon 
the disease. Chittenden 13 in England during 1908, 1909 and 1910 
carried out inoculation work with the supposed parasite, viz., Thie- 
lavia basicola, obtaining during the first two seasons negative results. 

In this work Chittenden 14 gave a good description of the disease. 
He found in 1910 that by excessive watering he was able to get infec- 
tions from Thielavia, but he did not indicate that they were the typ- 



ical "streak." He writes, "To sum up, as far as our experiments go, 
the "Streak" disease is brought about by the attack of the fungus 
Thielavia basicola on plants that have received some check at the root." 

During the season of 1912 Massee 15 again took up the disease, and 
attributed it to Thielavia basicola. 

Another worker in England, a Mr. Dyke 16 of St. Margarets found 
Macrosporium solani constantly associated with the disease, and be- 
lieved it to be the cause of the trouble. 

The serious nature of the disease may be seen by noting the num- 
erous popular articles which have appeared in various horticultural 
and gardeners' journals of England. Citations from several of the 
more important of these articles are as follows : 

In the Fruit-Grower, Fruiterer, Florist and Market Gardener, p. 
155 (England) of Sept. 5, 1907, Mr. T. A. Weston in the "Kent 
Notes" writes: 

"I regret to note how the stripe disease, Peronospora viciae, in- 
creases every year, and this season it has been very prevalent every- 
where. One always knows the tell-tale streaks in the foliage and vine, 
the stripes appearing in the flowers in due course. ' ' 

In the Oct. 24, 1907 number of the same journal Mr. Weston again 
writes upon the same disease, partly quoting a letter from a friend 
in Ireland. 

"Mr. B — has the same thing in freshly broken up grazing land, 
no manure of any kind being used. Haulm 7-8 in. wide on healthy 
plants on same ground." 

He goes on to say that he sent a couple of plants to Mr. Massee, of 
Kew, who gave it as Peronospora trifoliorum. That was two years 
ago. This year he has had an expert watching haulm and foliage 
under a microscope since the disease showed itself, the plants at that 
time being 6 in. Whole plants 6 ft. have been lifted and grown under 
conditions that would have brought fungoid disease out if it existed, 
yet no trace of such has been discovered either in the tissues or exter- 
nally. The roots of diseased plants all showed very poor root develop- 
ment, few fibres and total absence of nitrogenous nodules. Whole 
batches of some sorts were affected, while with others a plant here and 
there showed the effects. Some showed the trouble before a flower ap- 
peared, others gave some beautiful flowers and then went wrong. 

"So far, my friend. Now, this is as I myself find it. A lot of 



my plants are going off, and I am sending you samples of growth, roots 
and flowers. Is it an atmospheric disease, soil disease, eelworm, or 
carried over in the seed ? A friend of mine near London also reports 
an awful time this year, and he cannot get any really useful advice. 
I've seen journal experts referring to it as stated by Massee, but I am 
beginning to doubt it very much. My Irish friend has sprayed with 
copper without avail. Kent T. A. W. " 

In "The Gardener" for Nov. 16, 1907, p. 585, Mr. Weston gives 
a somewhat more detailed statement of the disease. The article is here 
quoted in full: 

THE STREAK DISEASE OF SWEET PEAS 

"I fully expected that this steadily increasing trouble would have 
been discussed by the many able exponents of Sweet Pea culture con- 
nected with The Gardener. 

"My note on p. 419 brought me several letters from a gentleman 
well known by name to the sweet pea world, and I venture to quote 
a few extracts, as they tend to show how serious the trouble is. The 
writer, an amateur by the way, says, 'I have had the disease in my 
garden for years, and it has been worse than ever this year, although 
most of my Sweet Pea plots have been replaced with fresh top spit 
loam from grazing land. I put 20 tons of this soil in a 60-feet row. 
A friend also broke up a new piece of pasture, giving nothing in the 
way of manure, not even artificials, and his plants were equally as 
bad as my own. Plants that escaped the disease were tremendously 
vigorous. I am of the opinion that the Peronospora theory is played 
out, for this year I had many plants carefully tested under the micro- 
scope, both root and branch, throughout the season, and not a trace of 
fungus was to be found. Some plants showed the disease, or what- 
ever it is, before any flowers developed, others gave a few blooms and 
then went wrong. I am so disheartened that I think of giving up 
Sweet Peas altogether.' 

"The foregoing tends to show that there is danger in this disease. 
As I have previously stated, I have seen the trouble for years, but 
never looked upon it as serious until two years ago, when it prevented 
my cutting any quantity of King Edward VII. This year I found 
it decidedly serious, more so when a gentleman visiting the National 
show told me that nearly all his plants were ruined by it. 

"With us two or three clumps showed the disease early in the 
season, while single plants in other clumps also gave evidence of the 



trouble, and so seriously were they affected that I pulled up the plants. 
One I particularly noticed, because it was the only one remaining of 
the variety. This showed a brown mark a few inches above the ground, 
the plant then being about 1 yard high. In a few days the brown 
mark had extended and had totally encircled the main stem, and also 
one of the laterals adjoining, while other laterals also showed signs 
of attack. The badly affected lateral went off limp, and on cutting it 
away I found the tissues, where affected by disease, quite dead. So 
alarmed was I that I scraped with a knife the outer covering of the 
main stem where affected, and could not find any live or green tissue, 
although the plant was still healthy. On close examination I found a 
plant of another variety similarly affected, and in utter hopelessness 
I painted the affected parts with strong Bordeaux mixture. The single 
plant got no worse, and eventually became a giant specimen, the other 
collapsed. 

"Later on came the streaked foliage amongst many plants, while 
streaked, starved looking flowers were produced from all affected 
plants. The disease did not confine itself to any one variety, nor to 
every plant of one variety. Nigger, Midnight, Lord Nelson, Hetty 
Green, Helen Pierce, Frank Dolby, Enchantress, John Ingman, Cod- 
sail Rose, and Lady Pollock all gave us streaked flowers, and although 
the affected plants continued to grow and flower right up to October, 
they never recovered normal health ; in fact, as the season advanced 
the flowers got worse, until it was impossible to find a flower worth a 
second glance. It has been declared to be the Vetch disease, Perono- 
spora trifoliorum, and sulphide of potassium, or sulphurated potas- 
sium has been given as the remedy. A local grower found this of no 
value whatever, whilst the writer of the letters referred to has tried 
everything possible without result ; and the fact of his being unable 
to discover disease spores in any shape or form shows it to be useless 
to use spray fluids. Another well known grower, however, informed 
me that after spraying with 'sul. potass.' his plants sent out new 
growth unaffected by the trouble. 

"A peculiarity about the diseased plants is their poor root de- 
velopment, nodules being totally absent. Personally I am doubtful 
as to the trouble being due to Peronospora. Unlike the Sweet Pea 
blight, the disease under notice does not confine itself to the foliage 
at the start, but seems to attack the whole plant at once. The stems 
show brown streaks, as do the leaves, whilst the tops of the growths 



are curled and twisted. The flowers are distorted and look quite out 
of character. Is the disease a product of California? Is it a bac- 
terial disease? Has it any affinity to curl disease in Potatoes? If it 
comes through the seed, why are not all the plants from one packet 
affected? Advice is urgently needed by growers both in England and 
Ireland. Who can aid us? T. A. W." 

In the February (1908) issue of the "Amateur Gardening" 
(England) p. 643, the "Streak" disease is again described as fol- 
lows : 

"STREAK IN SWEET PEAS" 

"Sweet pea growers in many parts of the country have had to 
contend with a comparatively new disease called the ' ' Streak, ' ' which 
threatens to considerably hamper the successful growth of this popu- 
lar flower. 

"This disease appears in the form of greyish or brownish streaks, 
either on the plants when young or when in flower. The effect of an 
attack is the loss of natural color in the foliage and a sickly diseased 
appearance. In some cases whole groups of rows of plants have been 
quite ruined by the disease. 

' ' So far its origin is more or less of a mystery, and all attempts 
to find a satisfactory remedy for it have resulted in failure. A pecul- 
iarity of the disease, apart from the streaks on the foliage, is the ab- 
sence on attacked plants of the usual bacterial nodules on the roots. 
This naturally lead^ to the inference that a cold and wet season like 
that of last year may have prevented the nitrogen-fixing bacteria doing 
their work. 

"The special appearance of the foliage caused by this disease 
must not be confounded with the grey and streaky results following 
an attack of thrips, or by sun scalds. The former is accompanied on 
the under side of the leaves by black shining dots ; the latter is only 
partial. The Peaman" 

In the Aug. 19th (1911) number of the "Amateur Gardening" 
the seriousness of the disease is clearly brought out as follows : 

"SWEET PEA DISEASE" 

"As stated in a recent note, the streak disease has once again put 
in an appearance, and I am fain to confess that it has proved more 
virulent and destructive than ever before. In a season like last, the 
disease was somewhat slow in accomplishing its deadly work; in fact 



9 

the agony was long drawn out, for the plants continued to live for 
varying lengths of time, although incapable of producing satisfactory 
flowers. This season, however, there has been no mistaking its action, 
and one can only assume that the great heat has materially assisted 
the disease to accomplish its purpose. During the eight years that I 
have been acquainted with this disease, I have never known it to work 
such havoc as it has done this season, and now that five-sixths of my 
plants are wiped out beyond recall I feel I must record a few impres- 
sions. 

"I do not for one moment suppose that I am relating a solitary 
case. More than one expert grower informed me that they were un- 
able to show at the National owing to the disease. One of these gen- 
tlemen has won championship cups in previous years, and he assured 
me his plants had collapsed by the cartload. This tale was told me 
at the Olympia Show. At the Sweet Pea Show, no less than six ex- 
pert gardeners from one district in Kent were upon my heels for a 
remedy. The disease was worse with them than ever before. At the 
sweet pea outing one or two efforts were made to secure some informa- 
tion, but the end of it all was nothing. 

"What does it mean? Does it mean total annihilation of sweet 
peas? Plants affected by the disease do not produce seed. The re- 
sults of my planting upon manured soil suggests that the disease comes 
to overfed plants, but the fact remains that streak can and does affect 
some plants on unmanured soil. It was visible at the N. S. P. S. 
trials, where no coarse growth is encouraged. I have seen it in the 
fields where sweet peas are growing for seed. It is not infrequently 
seen in America, for this much I elicited from one of our recent Ameri- 
can visitors." "The Peaman." 

Thruout the season of 1911 and 1912 discussions of a popular 
nature upon the "streak" disease of the sweet pea are found in many 
of the gardening and horticultural journals. The articles in the Ama- 
teur Gardening, The Gardeners' Chronicle and the Annual Reports of 
the National Sweet Pea Society, throw much light upon the nature of 
the disease. 

The writer 17 presented a paper illustrated by lantern slides before 
the American Phytopathological Society at Cleveland on Jan. 3 of 
this year giving a preliminary report on his findings about this dis- 
ease. The causal organism was announced as a newly described path- 
ogen under the name Bacillus lathyri. 



10 

Directly following the above paper, W. Bateson read a paper by 
Dorothy M. Cayley 18 before the Royal Society (London) under the 
caption "A Preliminary Note on a New Bacterial Disease of Pisum 
sativum" in which is described a disease having symptoms resembling 
in many respects those of the "streak" disease of sweet pea. 

I quote from her paper the following: 

' ' Investigations have been carried out this year at 'the John Innes 
Horticultural Institution to elucidate the nature of a disease which 
affects culinary peas (Pisum sativum). 

"The disease, in this district at all events, is a serious one, kill- 
ing a large proportion of the crop, but I have no information as to its 
prevalence in other parts of the country. I have succeeded in proving 
that the disease in culinary peas is caused by a large bacillus which 
exhibits a peculiar feature, inasmuch as it is transmitted in the inte- 
rior of the seeds of the plant. As far as I am aware no analogous in- 
stances are known. 

"The general symptoms are as follows: In mild cases after germ- 
ination the shoot can develop normally, but in bad cases it is frequently 
abortive, brown and dead at the tip, and laterals grow out prematurely 
to take the place of the main shoot. Quite early in the development of 
the plant, when the plumule is from half an inch and upwards in 
length, light brown longitudinal streaks can be seen on the stem and 
root, and the first leaves are often brown at the tip. These streaks 
develop later into slits. In very bad cases little or no germination 
takes place. After this stage no further definite signs are noticeable 
till about the flowering period. Then the development of the disease 
depends a good deal on external conditions. If the weather is warm 
and dry, and the plants are growing vigorously, the disease develops 
rapidly, and in a few days the plants become unhealthy and change 
colour. The stem turns slightly brown, and looks somewhat water- 
soaked. Brown longitudinal streaks appear at the base of the petioles 
on either side of the rib of the stem, which is continuous with the 
mid-rib of the leaf. The streaks split open and dry out. The collar 
may be badly disorganized. The leaves become spotted, streaked and 
yellowish in color, and if the disease is progressing rapidly the younger 
portions of the plant show discoloration, and fail to develop properly. 

"Except in bad cases the plants grow to full height, and can 
flower and set a certain amount of seed, but on examination the coty- 
ledons of the seeds of a diseased plant show brown discoloration, which 



11 

may be limited to a mere spot in the centre of each cotyledon, or, on 
the other hand, nearly the whole of the cotyledon may be involved. In 
the latter case there is often a cavity in the centre of the cotyledon. 

"Sections of the diseased cotyledon show large numbers of bacilli 
in various stages of development in the cells and intercellular spaces. 
' ' The bacillus works it way into the intercellular spaces and then 
breaks into the cells. There the nucleus is often attacked, the cyto- 
plasm destroyed, and the cells collapse, thus forming rents in the tis- 
sues . 

"There is considerable evidence to show that the bacillus passes 
up the plant through the tissues above mentioned, through the funicle, 
and probably the micropyle into the young developing seed. If one 
pea is diseased all the other peas in the same pod are diseased to an 
equal extent. The disease is chiefly spread by the seed, but fresh in- 
fection may take place through the soil. 

"Inoculation experiments were carried out in the open, but little 
stress can be laid on the results, as the disease w T as so prevalent 
throughout the experimental plot. Pea plants grown in heated soil 
in boxes, and inoculated just above the ground, when the plants were 
about 1 foot in height, showed no disease, whereas, in the open, seven 
out of ten inoculations on the stem just below the youngest unfolding 
leaf were successful. 

"Further inoculation experiments are necessary, but the above 
results tend to show that the bacillus can only penetrate very young 
tissue. This is supported by the fact that large numbers of the bacilli 
have been found in the inner tissues of the radicle when only about 
half an inch long. 

"Further investigations are in progress. 

"In many respects the symptoms resemble those of the formid- 
able disease of sweet pea (Lathyrus odoratus) known as "streak." 
This disease has been held to be due to Thielavia basicola, but, in view 
of these observations, that conclusion seems very doubtful, and I may 
add that, in the stem of diseased sweet peas, I have already found bac- 
teria like those here described." 

The fact that Miss Cayley has isolated bacteria from the stems 
of sweet pea affected with "streak" has caused her to question the 
conclusions of Massee and Chittenden, viz. that Thielavia basicola is 
the cause of "streak." 

Beginning with the issue of Apr. 5 (1913) of the Gardeners * 



12 

Chronicle, the writer and his associate Mr. J. J. Taubenhaus published 
several articles giving a review of the diseases of the sweet pea ( Lathy - 
rus odoratus) to date. 

The first article in the issue above noted describes and illustrates 
the "streak" disease. 

SYMPTOMS OF THE "STREAK" DISEASE 

On Sweet Pea. Like the Bacteriosis of beans, it makes its ap- 
pearance in the season of heavy dew. On the sweet pea the disease 
usually appears juct as the plants begin to blossom ; it is manifested 
by light reddish brown to dark brown spots and streaks (the older 
almost purple) along the stems, having its origin usually near the 
ground, indicating distribution by spattering rain and infection 
through the stomata (See PI. 3). The lesions which at first are sepa- 
rate and distinct soon confluently meet, causing the streaked appear- 
ance. The disease becomes quickly distributed over the more mature 
stems until the cambium and deeper tissues are destroyed in continuous 
areas when the plant prematurely dies. Occasionally petioles and 
leaves show infection, the latter show the watersoaked spots common 
to the bacterial leaf blight of beans. 

The disease is not a vascular infection ; it confines its attack to the 
mesophyll, the cambium and deeper parenchymous tissues ; the lesions 
on the stems gradually enlarge and deepen till they come together. On 
clovers (Trifolium spp.) the disease first appears in August and Sep- 
tember on the young seedling plants, when often it is particularly se- 
vere, vying with Bain's Anthracnoseo in its activity. In young clover 
it causes leaf spot (See Plates 7-12), water soaked as in the bean, and 
it also attacks the petioles and crown. In more mature clover prob- 
ably the most severe attack takes place in the petiole and sheath at the 
union with the stem; in this case the entire leaf dies and the lesion 
extends down into the stem. The blackening of the stems and the 
spotting and water soaking of areas on the leaves are common with 
the clovers (See Plates 6, 7, & 8). It is not uncommon to find the 
blackened lesions on the stems of clover so overlapped as to cause the 
entire stem to darken. 

Soy Bean. The disease was particularly severe upon one variety 
of soy bean, in which the lower lesions girdled the stein or penetrated 
so deeply that the plants blew over, suggesting the black leg of pota- 
toes (See Plates 13, 14, and 15). The lesions were common on the 



13 

upper branches and even the pods, the latter showing very conspicu- 
ous blackened spots (Plate 14). 

Bean (Phaseolus spp.) On several varieties of beans a stem 
lesion, which was supposed to be caused by Psendomonas phaseoli, 
showed upon culturing an organism similar to that met with in the 
sweet peas, the clovers, and the soy beans. These lesions usually were 
small elongated, rusty brown areas one-fourth to several inches in 
length; occasionally the lesion was slightly sunken (PL IV). 

In order to compare this organism with that of Pseudomonas 
phaseoli of bean blight some 350 isolation plates were made from be- 
ginning lesions on bean stem and leaves. Instead of Ps. phaseoli in 
the stem lesions, in most cases this new organism was found. From 
watersoaked pods and leaves a Pseudomonas was obtained which an- 
swered closely to Ps. phaseoli (PI. 5). 

In order to determine the position of our work, extensive isola- 
tions, cross infections, and systematic studies were started. 

ISOLATION AND MORPHOLOGICAL STUDIES 

Over 1500 plate cultures of beginning or young lesions were made 
from the several hosts. The organism may be taken almost invariably 
in abundance in pure culture from the beginning lesions in the stems 
of sweet peas when the surface is properly sterilized. Some difficulty 
was experienced at first in taking the organism from the clovers. This 
we attributed to non selection of young lesions, to too severe surface 
sterilization and to the ease with which the lesions in the thin cambium 
of the clover dry out, thereby causing the death of the organism. 

The isolation work clearly indicated that the parasite was bac- 
terial; a yellow organism which grows luxuriantly upon all the nut- 
rient media and especially rapid upon nutrient media containing 
sugars. On standard nutrient glucose agar the colonies appear within 
24 to 36 hours. The center becomes granular and the colonies have a 
marked tendency to become stellate or auriculate. 

Morphological studies showed the organism to be a comparatively 
small rod-shaped bacillus, which in fresh cultures is never found in 
chains, and seldom even united in twos or fours. The flagella are not 
easily demonstrated. (See Fig. 1). 



14 

INOCULATION EXPERIMENTS 

Preliminary inoculation work was carried out Aug. 9, 1912 with 
sweet peas using two cultures, viz. sources No. 1 and No. 3 (see p. 17). 
The former came from sick sweet peas at the Experiment Station farm 
at Newark, Del., and the latter from sweet peas affected with "streak" 
from Boston, Mass. Inoculations were carried out by atomizer spray- 
ings with 48 hour cultures, after having first thoroughly wet the 
plants. The sprayings were applied in the evening on all parts of the 
plants above ground. Typical "streak" infections showed on the 9th 
day. On the 10th day lesions were cut out, surfaces sterilized, washed, 
crushed, and cultured with the result that the typical organism used 
in the inoculation work was recovered. Inoculations were repeated on 
Aug. 22, 1912, this time using cultures of source No. 8 from sweet peas 
affected with "streak" sent here by T. A. Weston from Orpington, 
England, and source No. 13a from Red Clover. The inoculations were 
this time both sprayed on certain plants with atomizer, while others 
were pricked with hypodermic needle. In both cases, infection was 
obtained in from 7 to 10 days. The spots pricked by the needle were 
the first to show infection, these appearing in seven days. The red 
clover cultures gave equally strong infections as did the sweet pea. 
Subsequent platings of these infections gave an organism identical 
with that used in the inoculation work! 

Several of the sources used in the cultural and biological studies 
were those secured from this preliminary infection work, viz. cultures 
No. 4, No. 5, and No. 7 and No. 10 E. 

Following the above preliminary inoculation experiments a series 
of cross infection work (Sept. 5) was tried out in the field with only 
partial success. Tall moist chambers were placed over small young 
plants of clover, alfalfa, cowpeas and soybeans, using cultures from 
sweet pea and from red clover. The red clover showed infections from 
the bacteria taken from Lathyrus spp. while, on the other hand the 
cowpea, soy bean and alfalfa showed no infection whatever. On Sept. 
15, this series was again tried out with only evidence of infection on 
the red clover. It would seem that alfalfa, and cowpea as well as soy 
bean in the young stages are not readily susceptible to this parasite. 

CROSS INOCULATION IN THE LABORATORY 

About the first of October (1912) some two dozen red clover 
plants were transplanted in eight inch pots and taken into the labora- 
tory where they made excellent growth. All evidence of infection was- 



15 



removed and the leaves were thoroughly sprayed with potassium per- 
manganate to surface sterilize them; the disinfectant was later care- 
fully washed off; the clover plants were then placed under moist jars 
for several days to note whether any natural infection would follow. 
The sterile plants were then thoroughly covered by atomizer, first with 
sterile water and then sprayed with young cultures of the streak dis- 
ease bacterium from ten different sources, viz., one from red clover 
and one from alsike clover, two from sweet pea, one from another 
Lathyrus sp., two from soy bean, one from Lima bean, one from al- 
falfa, and one from infected soil. 

The following table shows the amount of infection on the clover 
leaves and stems. 

Table Showing Amount of Artificial Infection on Red Clover 



1. Red Clover on Red Clover 


10% of infection 


2. Sweet Pea (American) on Red Clover 


12% < 


< i 


3. Lathyrus sp. " " " 


99% < 


c i 


4. Soy Bean " " 


4% ' 


1 1 


5. Soy Bean " " " 


60% ' 


1 1 


C Sweet Pea (English) " " 


50% ' 


i i 


7. Alsike " " 


75% ' 


( c 


S. Lima Bean 


40% ' 


I ( 


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The results of these infections are shown in Plates 10, 11 and 12. 
Infection began in 3 to 7 days. The leaf lesion is a typical water- 
soaked area, quite similar to that of the bean blight disease. 

These experiments were carried out again beginning Nov. 11, 1912, 
along with some additional inoculations. Pseudomonas phaseoli of 
bean blight was tried on red clover but it failed to produce any infec- 
tion. Three sources of the "streak" disease organisms were used from 
sweet pea,' and two sources from forage species of Lathyrus. Infec- 
tion was evident in three days. Two sources of the "streak" organism 
B. lathyri from beans were used getting definite infection in three to 
five days. From this work it was quite apparent that the organism 
from the several different hosts was capable of producing the disease 
on red clover identical with that in the field. 

Many different attempts were made to get infection by spraying 
on clover and sweet peas and not paying any attention to keeping the 



16 

plants moist. Invariably the results were negative. All attempts to 
infect young sweet pea plants have been failures. The disease will 
not develop till the plant is about to blossom. Just what physiological 
changes take place in the plant just prior to and at the time of blos- 
soming has not been determined, but it is evident that there must be 
a radical change, as needle inoculations or sprayings will make no ad- 
vance whatever. 

The writer is of the opinion that along with a possible change in 
the physiology of the plant, owing to the fact that the flowering period 
falls in the time of heavy dew, there is a turgescence in the plant 
throughout the nights which probably favors the entrance and growth 
of the organism into the interior of the plant. 

THE RELATIONSHIP OF THE CASUAL ORGANISMS 

The series of apparently similar organisms taken from such a va- 
riety of hosts offered a splendid opportunity for studying the possible 
cultural and biochemical variation. Having proved that red clover 
could be infected with the organisms from the different sources (see 
p. 14) these same sources, together with several morphologically sim- 
ilar organisms from other hosts were used for a detailed study of 
variation in cultural, physical and bio-chemical features. In order to 
be certain that the media were standard in their properties and reac- 
tions, several well known organisms were run as checks. For this pur- 
pose Bacillus coli, Pseudomonas compestris and Pseudomonas phaseoli 
were selected. The cultures used in this study were taken from begin- 
ning lesions which appeared free from contamination. 

The isolation of bacteria from lesions was carried out by following 
a method which has been used by us 21 for a number of years. 

This consists in cutting out the lesions somewhat beyond the area 
of infection, pieces being small enough to be placed in a culture tube. 
If the lesions are on small stems, as that of clover or sweet pea, sec- 
tions of the stem, including lesions are cut out one-half to three-fourths 
of an inch long. If the lesions are on the leaf, the youngest infections 
are selected and taken out entire. The surface sterilization is carried 
out in a clean test tube, upon several pieces at a time, using enough of a 
50% alcoholic solution containing one gram of bichloride of mercury 
to the liter, to cover the infected pieces of plant tissue at a depth of one 
inch. After placing the plug in the tube and shaking the disinfectant 
to all parts of it, the solution is allowed to stand on the material from 
15 seconds to two minutes, depending on the nature of the plant tis- 



17 

sue. Thin leaves, lightly infected, will hardly stand more than 10 
seconds, while deeply infected stems and tubers, or root tissues will 
stand two minutes, or even much more when the disinfectant does not 
penetrate too deep. At the required time, the disinfectant is poured 
off and the material is washed three times with 12 to. 15 cc. of sterile 
water for each washing. This washing is carried out in the same tube 
in which the surface sterilization is done. Sterilized water for this 
purpose is kept in stock at all times in the laboratory, stored in tubes 
containing 12 to 15 cc. After flaming the plug and mouth of the tube 
carrying sterile water, the contents is poured directly upon the mate- 
rial to be washed and the flamed plug is used to close the tube. The 
water is thoroughly shaken to all parts of the tube in order to wash 
away every trace of the disinfectant. This water is then poured off 
and the process repeated, each time using the new cotton plug from 
the sterile water tube, until three washings have been given, when 
the material is gathered near the mouth of the tube. Here the indi- 
vidual pieces are picked out with sterile forceps and each is crushed 
with the same forceps in the mouth of a tube containing a medium 
properly cooled for growth. The crushed lesion is washed down by 
the medium, sub-cultures are made and poured into sterile' plates. 

'The writer has had excellent success with this method, succeed- 
ing almost invariably in taking out pure cultures of the pathogen, 
whenever it is not associated deeply in the lesion with contaminating 
organisms. 

The following is a list of the organisms isolated and used in the 
cultural studies : 

No. 1. From Sweet Pea Stem, showing typical "streak" obtained 

July 20, 1912 from the Experiment Station farm at New- 
ark, Del. 

No. 2. From Sweet Pea Stem showing typical "streak" obtained 
July 25, 1912 from the Experiment Station farm at New- 
ark, Del. 

No. 3. From Sweet Pea Stem showing typical "streak" obtained 
Aug. 1, 1912 from the noted sweet pea grower, Mr. William 
Sim of Boston, Mass. 

No. 4. From Sweet Pea Stem showing typical "streak," artificial 
infection in garden of T. F. Manns, Newark, Del., Aug. 18, 
1912. 



18 

No. 5. From Sweet Pea Stem showing typical "streak" by arti- 
ficial infection in garden of T. F. Manns, Newark, Dei., Aug. 
18, 1912. 

No. 7. From Sweet Pea Stem showing typical "streak" by arti- 
ficial infection in garden of T. F. Manns, Newark, Del., Aug. 
21, 1912. 

No. 8. From Sweet Pea Stem showing typical "streak" obtained 
Aug. 22, 1912. from T. A. Weston, St. Johns Road, Orping- 
ton, England. 

No. 9. From Sweet Pea Stem showing typical "streak" obtained 
Aug. 24, 1912 from T. A. Weston, St. Johns Road, Orping- 
ton, Eng. 

No. 10 e. From Sweet Pea Stem showing typical "streak" from ar- 
tificial infection Aug. 22, 1912. from T. F. Manns' garden, 
Newark, Del. 

No. 12 d' From Sweet Pea Stem showing typical "streak" obtained 
from J. J. Taubenhaus' garden, Newark, Del., Sept. 24, 
1912. 

No. 12. h'. From Sweet Pea Stem showing typical "streak" obtained 
from J. J. Taubenhaus' garden, Newark, Del, Sept. 24, 
1912. 

No. 43 c. From Sweet Pea Stem, showing typical "streak" obtained 
from Marshall Manns' Garden, Oct, 25, 1912, Newark, Del. 

No. 13 a. From Red Clover petiole, showing black lesion in cambium, 
obtained Sept. 4, 1912 from the Experiment Station Farm, 
Newark, Del. 

No. 14 i '. From Red Clover Stem, showing darkened lesion in cam- 
bium, obtained Sept. 7, 1912 in vicinity of Newark, Del. 

No. 15. From Red Clover Stem, showing black lesion in cambium, 
from field near Mr. Taubenhaus' home, Newark, Del., Sept. 
24th, 1912. 

No. 16 i. Same source as No. 15. Different plant. 

No. 41 K. From Red Clover Stem showing black lesion in cambium, 
called "OK Culture" becaase material so typical, from 
vicinity of Newark, Del, Sept. 25, 1912. 



19 

No. 17 d'. From Alsike Clover petiole, showing typical blackened le- 
sion, from field near Red Men's Home, Newark, Del., Sept. 
4, 1912. 

No. 18 e '. From Alsike Clover petiole, same origin and date as No. 
17 d' though different plant. 

No. 21. From Soy Bean petiole, showing small sunken black lesion, 
from Experiment Station farm, Newark, Del., Sept. 3, 1912. 

No. 22. From Soy Bean Stem, showing beginning black lesion, from 
Experiment Station farm, Newark, Del., Sept. 7, 1912. 

No. 23 h'. From Soy Bean Stem, showing beginning lesion, from Ex- 
periment Station farm, Newark, Del., Sept. 24, 1912. 

No. 24 h '. From Soy Bean stem, from beginning black lesion, same 
source and date as No. 23 h, though different plant. 

No. 25 d 2 . From Lathyrus Sp. (for forage purposes) showing typical 
"streak" lesions on stem, obtained from Experiment Sta- 
tion farm, Newark, Del., Sept. 3, 1912. 

No. 26 e'. From Lathyrus Sp. (for forage purposes) showing typical 
"streak" lesions on stem, obtained from Experiment Sta- 
tion farm, Newark, Del., Sept. 27, 1912. 

No. 26 f '. From Lathyrus Sp. Same source and date as No. 26 e' only 
a different plant. 

No. 28. From Cowpea Leaf, sent in from vicinity of Philadelphia, 
leaves turning brown : Aug. 3, 1912. 

No. 30. From Wax Bean seed taken from green pod showing large 
watersoaked lesion; obtained from Red Men's Home, New- 
ark, Del., Sept. 3, 1912. 

No. 36. From Tomato fruit showing typical end rot of fruit, from 
T. F. Manns' garden, Newark, Del, July 13, 1912. 

No. 37. From Tomato fruit end rot produced artificially by inocula- 
tion with source 26 above, from infection in laboratory, 
Newark, Del. Variety of Tomato "Stone," July 28, 1912. 

No. 40 g 2 . From Climbing Bean pod (green string bean). Typical 
brown sunken lesions along pod; from Mr. Taubenhaus' 
garden, Newark, Del., Sept. 30, 1912. 



20 

No. 42. From Dwarf Bean, watersoaked pod, plate No. 15, from 
Mr. Taubenhaus' garden, Newark, Del., Oct. 27, 1912. This 
organism is Ps. phaseoli and was hence used as a check. 

No. 45. From Dwarf Bean Pod. Dark brown lesions along pod ; 
from Mr. Taubenhaus' garden, Newark, Del, Oct. 27, 1912. 
This organism is also Ps. phaseoli. 

No. 46. From Dwarf Bean Pod. Typically watersoaked lesion from 
Mr. Taubenhaus' garden, Newark, Del., Oct. 27, 1912. 
This organism is also Ps. phaseoli and used as a check. 

No. 47. Bacillus coli from human intestine. Used here as a stand- 
ard check on media and for reactions. 

CULTURAL AND BIOCHEMICAL STUDIES 

Extensive cultural and biochemical studies made upon the thirty- 
two different strains from the several hosts confirm the results from 
inoculation work, viz. that the causal organisms are identical as far 
as our present system of classification is concerned. Some slight vari- 
ations were noticeable in several of the strains, such as absence of 
pellicle in nutrient broth, or a very meagre pellicle. These differences 
were just as marked in the different strains from the one host as they 
were between the strains from the several hosts. This was likewise 
true for color gradations on the different solid media. The shades 
of yellow varied from a light straw to that of almost a deep orange. 
Probably the most uniform cultural reaction was that on nutrient 
gelatin, in which in practically every strain it required somewhat 
over two months for complete liquifaction, while at the end of six 
weeks not more than half of the gelatin was liquified. The fact that 
liquifaction proceeded entirely from above would indicate that the or- 
ganism is an obligate aerobe. 

In table I is shown in a brief summary the group number and 
other features of the thirty-seven organisms compared thruout the ' 
cultural studies. It is quite apparent that of the 32 strains first con- 
sidered in this summary and selected from various hosts because of 
similarity in disease production, or because of similarity in morph- 
ological and preliminary cultural studies, that there is not variation 
enough to differentiate one strain from another on any specific cult- 
ural reaction (See Table I). 



The introduction into this series of cultures for compaiativel 
studies of sources Nos. 36 and 37 from "point" or "fruit rot" of to- 
mato was to learn the cultural and other classifactory features of the 
organism which here is shown to be the active causal agent of point 
rot. (see PL 16). As far as the present system of bacterial classi- 
fication is applicable, this organism is not to be distinguished from 
that described herein as Bacillus lathyn n. sp. The writer did not 
carry out cross inoculation work on sweet pea and clover with the or- 
ganism from the point rot of tomato. Though the morphological and 
cultural features of the tomato organism are similar as far as we are 
able to determine by present methods, yet it is quite possible that the 
organism may not be able to cause the "streak" disease on sweet pea 
and other legumes. 

A short history of the association of bacteria with the "fruit rot" 
or ' ' blossom end rot ' ' of tomato is as follows : 

Elizabeth H. Smith 22 (1905) and F. S. Earle 43 (1900) have found 
bacteria associated with the fruit rot of the tomato. Both claim typ- 
ical infections when inoculations were made from pure isolated cult- 
ures. William A. Stuart 24 has also found bacteria associated with 
the fruit rot of the tomato. The writer in July 1912 found the rot 
quite general in his garden, and having seen Miss Smith's report of a 
"bacterial rot" of the tomato took the opportunity of culturing young 
lesions from affected fruit. Careful surface sterilization was prac- 
ticed ; only beginning lesions were cultured in the first series and the 
extreme margins of the lesions were used. The result was that in 
every plate a yellow bacterium came out. In several of the plates 
there were associated occasionally a Fusarium and an Alternaria. 
Both of these fungi as well as the bacterium were isolated in pure 
culture- on an artificial medium. Inoculation work was carried out 
with each of the organisms. The bacterium gave excellent infection 
both in ths field and in the laboratory during a period of dry weather, 
when sprayed with atomizer or inoculated by needle prick. During 
the same time there was also some evidence of infection when the Fu- 
sarium was inoculated into the flower end of the fruit by inserting 
mycelium beneath the epidermis with a sterile scalpel. The Alter- 
naria, however, made no progress at all. The check sprayings with 
sterile water and the check incisions gave no infections. The above 
experiments were duplicated during a period of rainy, cloudy weather 
with very little progress whatever in the production of rot. The Fu- 



22 




Fig. 1 



23 

sarium and Alternaria showed no evidence of infection, while the bac- 
terium produced only small watery lesions. 

Drought is a great factor in furthering the disease. Heavy rains 
with moist atmosphere entirely checked the disease in the experimental 
work. The writer is inclined to infer that this disease is of bacterial 
origin and that its progress is very closely associated with the water 
supply of the plant. 

(Since writing the above in June 1913 the writer has been privil- 
eged to review Brooks' work on the "Blossom-end Rot of Tomatoes," 
Phytopathology Vol. VI, No. 5, Oct. 1914. He still holds the view that 
the disease is of bacterial origin, and is distributed by rain or insects 
at blossoming time. The very beginning lesions give pure cultures 
of the bacterium.) 

DESCRIPTION OF THE CAUSAL ORGANISM* 

Bacillus lathy ri n. spp. Manns & Taubenhaus 

I. Morphology 

1. Vegetative Cells. When grown upon nutrient agar + !or 24 
hours at 25° to 28° C and stained with aqueous solutions of methylene 
blue, gentian violet or fuchsin the organism is shown to be a compar- 
atively small rod-shaped bacillus, with rounded ends which is rarely 
found in two or fours. The stain in the organism from such cultures 
is evenly distributed thruout the cytoplasm. In older cultures, two 
to three months, some take a denser polar stain. The organism mea- 
sures from .75" to 1.5" X .6 U to .85" the majority 'being 1.4" X .75". 

2. Sporangia. No sporangia have been observed. 

3. Endospores. Cultures on various media carried for ten months 
show no endospores. 

4. Flagella. When stained by Loeffler's, Pitfield's or Van Er- 
mengen's methods the flag^la may be demonstrated, though not easily. 
They are shed so readily that usually no more than from two to five 
may be shown attached, though it is not uncommon to find many de- 
tached thruout the field. However, when the material is carefully 
selected, fixed and stained, the flagella m ay 'be demonstrated to be well 
distributed peritrichially, and to number eight or even more. (See 
Fig. 1). 



*The thirty-seven descriptive charts used in tabulating the morphological, cult- 
ural, physical and biochemical features of the different organisms, are not printed 
herewith. 



24 



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26 

In hanging drop or under cover slip the organism in 24 to 48 
hour cultures is very active, taking a motion somewhat slower but 
otherwise similar to that of B. typhosus. 

5. Capsules. No capsules have been demonstrated. 

C. Zoogloea. No zoogloea has been formed on the ordinary media, 
but in asparagin broth, and in Uschinsky's solution a ropiness is com- 
mon; on starch jelly containing Uschinsky's solution a pseudozoog- 
loea is present. 

7. Involution forms. Individuals somewhat longer and broader 
than usual are met with in old cultures four to six months; bipolar 
staining, and denser regions of cytoplasm are commonly seen with 
ordinary stains. Some individuals show granulation, though this is 
not common. 

8. Staining reactions. The organism, though staining readily 
with the ordinary stains, loses these quickly when washed with alcohol. 

9. Gram's Stain. The organism is gram negative. 

10. Loeffler's Methylene Blue. With Loeffler's Methylene blue 
the cytoplasm shows no granulation and is evenly stained thruout from 
a 24 hour culture. 

11. Neisser's Spore Stain. No evidence of spores present with 
Neissers' stain. 

II. Cultural Features 

1. Agar Stroke — Nutrient Agar. 

Growth, at 25 to 28° rapid in 24 hours, and abundant 
in 72 hours. No growth at 37° C. 

Form of Growth, filiform, usually smooth at margin 
but occasionally undulate, or even slightly echinulate. 

Elevation of Growth, slightly convex. 

Luster, glistening. 

Topography, usually smooth ; surface occasionally very 
slightly granulate. 

Optical Characters, opaque. 

Chromogenesis, light to deeper yellow in different strains. 

Odor, absent. 

Consistency, butyrous. 

Sub medium, shows no change of color. 



27 

2. Potato. 

Growth, quite rapid in 48 hours. 

Form of Growth, filiform becoming more or less irreg- 
ular. 

Elevation of Growth, slightly convex. 

Luster, glistening. 

Topography, smooth. 

Chromogenesis, at first a light yellow, later becoming 
somewhat deeper. 

Odor, absent. 

Consistency, butyrous and slightly viscid. 

Medium, not changed. 

3. Loeffler's Blood Serum. 

(not used) 

4. Agar Stab. 

Growth, best at top-surface growth quite rapid in 48 
hours, abundant after 3 days. Organism a rather closely 
restricted aerobe. 

Line of Puncture, filiform, but quite restricted, in growth 
deep in the medium. 

Chromogenesis, yellow. 

Medium, not changed in color. 

5. Gelatin Stab, 

Growth, best at top, small amount of growth in the lower 
part of stab. 

Line of Puncture, filiform. 

Liquifaction, slow, not showing till nearly two weeks 
old and at end of four weeks only fairly well begun. Not 
complete till three months. Quite uniform thruout 32 strains. 

Medium, not discolored. 

6. Nutrient Broth. 

Surface Growth, slight pellicle in some cases though not 
general. 

Clouding, strong in 24 hours. 

Odor, absent. 

Sediment, compact, scant. 



28 

7. Plain milk... No visible change takes place in two weeks. At 
the end of three weeks 23 out of the 32 strains showed curdling with- 
out separation of whey or of digestion of curd, while 9 strains did 
not show coagulation until heat was applied. Tests showed that the 
acidity in the various strains which coagulated milk in three weeks 
gradually increased from Fuller's scale to -)-6 at end of the third 
day; to -4-12 at end of first week, and at time of coagulation, that is 
three weeks, had reached +36, Fuller's scale. It seems probable that 
where coagulation took place before evidence of peptonization of 
casein set in, that the increased acidity may have in some strains 
checked the growth of the organism. In the 9 strains noted above 
some evidence of digestion as well as acid production was showing 
though the acidity was not great enough to bring about coagulation. 
At the end of seven weeks 24 strains showed partial or complete di- 
gestion of casein, while eight remained curded with little or no evi- 
dence of digestion. In several strains there was evidence of curding 
from enzymatic action ; that is, the production of acid did not appear 
strong enough to bring about coagulation. 

8. Litmus Milk. 

In most strains there was a gradual and slow increase 
in acidity thruout the first month. In several of the more 
rapid digesters of casein the litmus was also digested and re- 
duced. 

9. Gelatin Colonies. Eight days old. 

Growth, medium. 

Form, round. 

Elevation, slightly convex. 

Edge, smooth. 

Liquif action, too slow to show on plate. 

10. Agar Colonies, five days old. 

Growth, rapid at 23 to 28° C. — Yellow colonies, visible 
to the eye in 24 hours. 

Form, stellate to ameboid. 

Surface, smooth, glistening. 

Elevation, slightly raised. 

Edge, entire and regular. 

Internal Structure, granular at center and smooth else- 
where. 



2!) 

Ch romogenesis, yellow. 

Size, depends on room in plate, much extended. 

11. Glycerine agar. 

Growth, rapid and abundant at 23 to 28° C. 

Form of Growth, filiform. 

Elevation of Growth, slightly raised. 

Luster, glistening. 

Topography, smooth. 

Optical charactei s, opaque. 

Ch romogenesis, light yellow. 

Odor, none, or slightly sour. 

Medium, not changed in appearance. 

12. Synthetic agar low in nitrogen. 

Weak to no growth. 

13. Cohn's solution at 23 to 28° C. 

Growth, absent. 

14. Uschinsky's solution at 23 to 28° C. 

Growth, rapid in 24 hours. 

Fluid, viscid yellow sediment after three days; some- 
times a pellicle would form and sink, followed by others. No 
fluorescence. 

15. Dunham's solution at 23 to 28° C. 

Growth, moderate. 

Clouding, moderat , persistent, fluid, slightly turbid. 
Indol formed. 

16. Asparagin solution at 23 to 28° C. 

Growth, abundant. 

Clouding, moderate, persistent, fluid turbid. 

17. Nitrate Broth at 23 to 28° C. ' 

Growth, moderate. 

Clouding, moderate, persistent, fluid slightly turbid. 

Nitrates, not reduced. 

18. Dextrose Bouillon at 23 to 28° C. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Gradually 
becomes acid from +7 Fuller's scale to +14 in 7 days. 



30 

19. Saccharose Bouillon at 23 to 28° C. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Gradually 
increases in acidity from +3 Fuller's scale to +9 in 7 days. 

20. Lactose Bouillon at 23 to 28° C. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Increases in 
acidity. 

21. Maltose Bouillon. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Increases in 
acidity. 

22. Glycerine Bouillon at 23 to 28° C. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Increases in 
acidity. 

23. Mannite Bouillon at 23 to 28° C. 

Growth, abundant. 

Clouding, strong, persistent, fluid turbid. Increases in 
acidity. 

24. Growth on Bouillon over Chloroform. 

Growth, absent. 

25. Sodium Chloride in Bouillon. 

4% inhibited growth. 

TABLE II-A. 

2. Production of Acid and Alkali (Fullers Scale) 

Degrees of Reaction after 
3 days 5th day 7th day 

+10 +12 +14 

+ 6 +8 +9 

— — Increased 



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days 


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+3 


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32 



26. Nitrogen, apparently is not obtained from the atmosphere, but 

is obtained from all the broths. 

27. Best medium for long continued growth. 

Nutrient glucose agar has given the longest growth in 
a shake deep tube culture. 

III. Physical and Biochemical Features. 

1. Gas production. No gas is produced from dextrose, sacchar- 
ose, lactose, maltose, glycerine and mannite broth in fermentation 
tubes, very little or no growth takes place in the arm. The limitation 
of growth was sharply defined at the union of bulb and arm, indicat- 
ing an obligate aerobe. 

3. Production of Ammonia. The production of ammonia has been 
determined in nutrient broth, Dunham's peptone solution, asparagin 
solution and nitrate broth. In all of these solutions some ammonia 
was present. The ammonification was quantitatively measured in a 
1% peptone solution. The following table (III) shows the amount 
of N/10 ammonia formed in 100 cc. of above peptone solution in seven 
days and twelve days respectively by eight different strains of Bacil- 
lus lathxjii from the following hosts; — sweet pea (3), Red Clover (2), 
alsike clover (1), soy bean (1), and wax bean (1). 

TABLE III 
Showing Ammonification 





In Seven Days 


In 


Twelve Da 


>'S 


No. of Culture and 
Host 


Total [J, 
NH 3 


V 

NH 
10 ^ j 

in check 


10 "3 

produced 


Total ^ 
NH, 


N NH 

10 3 

in check 


N N H3 

produced 


43 C. Sweet Pea 


9.1 cc 


1.6 cc 


7.5 cc 


1 14.6 cc 


1.9 cc 


17.7 cc 


9d 2 " " 


7.2" 


1.6" 


5.6" 


1 13.2" 


1.9" 


11.31" 


10 e " " 


7.2" 


1.6" 


5.6" 


13.2" 


1.9" 


11.3" 


13 a Red Clover 


6.7" 


1.6" 


5.1" 


11.5" 


1.9" 


9.6" 


14 i " 


3.9" 


1.6" 


2.3" 


10.9" 


1.9" 


9.0" 


18 Alsike Clover 


6.0" 


1.6" 


4.4" 


14.2" 


1.9" 


12.3" 


21 Soy Bean 


7.9" 


1.6" 


6.0" 


13.8" 


1.9" 


11.8" 


40g 2 Wax Bean 


6.1" 


1.6" 


4.5" 


12.4" 


1.9" 


10.5" 


Soil inoculation 


58.1" 


1.6" 


56.5" 


74.8" 


1.9" 


72.9" 


Check Culture 


1.6" 


1.6" 


1.0" 


1.9" 


1.9" 


0.0" 



4. Nitrates in Nitrate Broth. No nitrate reduction takes place. 
Several of the strains produce nitrites from peptone broth ; thus in 
nitrate broth unless quantitative determination is made for the nitrates 



33 



placed therein one might easily assume nitrate reduction, 
is not formed from nitrates. 



Ammonia 









TABLE IV 
















Showing Growth on Acid and Alkali Media. 






Culture and Host 




-10 





+15 


+25 


+35* 


1 


B. 


lathyri, Sweet pea 


+ 


+ 


+ 








2 


* * 


c < < i 


1 1 


+ 


+ 


+ 








3 


i i 


a a 


1 1 


+ 


+ 


+ 








4 


i '. 


(i t i 


1 1 


+ 


+ 


+ 








5 


' ' 


ll 


1 1 


+ 


+ 


+ 








7f 




. . ( < 


t i 


+ 


+ 


+ 








% 


' ' 


if 


1 1 


+ 


+ 


+ 





o. 


9d 2 


i i 


( i t i 


1 1 


+ 


+ 


+ 








lOe 


i i 


a a 


1 1 


+ 


+ 


+ 








12d' 


i i 


tt i i 


1 1 


+ 


+ 


+ 








12h' 


11 


tt i i 


1 1 


+ 


+ 


+ 





0' 


43c 


1 < 


i i it 


1 1 


+ 


+ 


+ 








13a 


1 1 


" Red 


Clover 


+ 


+ 


+ 








14i 


" 


. i a 


1 1 


+ 


+ 


+ 








16c 


< i 


it it 


1 1 


+ 


+ 


+ 








15 


i i 


i ( i i 


1 1 


+ 


+ 


+ 








16c 


i i. 


( ( i i 


1 1 


+ 


+ 


+ 








16i 


1 < 


(i i i 


1 1 


+ 


+ 


+ 








, -; , : 


t i 


a a 


1 1 


+ 


+ 


+ 








17 


1 1 


" Alsike Clover 


+ 


+ 


+ 








18 


< < 


i i i i 


1 1 


+ 


+ 


+ 








21 


i i 


" Soy Bean 


+ 


+ 


+ 








22 


i i 


i i ( i 


1 1 


+ 


+ 


+ 








23h ' 


< i 


a t c 


1 1 


+ 


+ 


+ 


' 





2'ir 


i i 


a it 


1 1 


+ 


+ 


+ 








25 cl„ 


1 1 


' ' Lath} 


r rus spp. 


+ 


+ 


+ 








26e 


i t 


it a 


1 1 


+ 


+ 


+ 








26f 


t i 


it it 


1 1 


+ 


+ 


+ 








28 


1 1 


" Cow 


pea 


+ 


+ 


+ 








30 


i i 


" Wax 


Bean pod 


+ 


+ 


+ 








36 


1 1 


" Tomato End Rot 


+ 


+ 


+ 








37 


( i 


a a 


it 1 1 


+ 


+ 


+ 








40g 2 


i i 


" Climbing Bean Pod 


+ 


+ 


+ 








-L2 


Ps 


phaseoli Dw 


arf Bean Pod 


+ 


+ 


+ 








44 


Ps 


campestris Black Rot Cabbage 


+ 


+ 


+ 








45 


Ps 


phaseoli, Dw 


arf Bean Pod 


+ 


+ 


+ 








4- 


Ps 


phaseoli. Dwarf Bean Pod 


+ 


+ 


+ 








17 


Ba 


cillus coli. 




weak 


+ 


+ 









5. Indol Production. Indol was produced in each of the 32 
strains tested out. In several the reaction for indol was light while 



34 



in the majority it was nearly equal to that in the eh. 
Bacillus coli. 



culture, viz. 



6. Relation to acid and alkali. The 32 strains of Bacillus lathyri 
together with 3 strains of Ps. phaseoli, one strain of Ps. campestris 
and one strain of Bacillus coli were tested on a broth medium varying 
in acidity and alkalinity. Table IV shows growth ( + ) and absence 
of growth ( — ) on media of folio wing reactions — 10, 0, -j-15, +25, 
and —(—35 Fuller's scale. 



7. Optimum Reaction. 
+10 Fuller's Scale. 



The optimum reaction is between and 



8. Vitality on Culture Media. Vitality on culture media is quite 
prolonged. Eight months' old shake cultures on deep, moist nutrient 
giucose agar gave strong growth when transferred to plate dilutions 
and to slant cultures. 

9. Temperature Relations. — Thermal Death Point, determined by 
heating tubes containing 10 cc. of broth to the desired temperature, 
then inoculating with loops of fresh culture in bouillon, al- 
lowing the inoculated tube to remain in water at same temperature 
for 10 minutes, then removing and cooling. Cloudiness within ten 
days indicated growth. The thermal death point is between 48° C 
and 50° C, 

The following table shows the thermal death point for five sources 
of B. lathyri and three sources of the checks viz. Ps. phaseoli, Ps. 
Campestris and B. coli.. Each was run in duplicate and incubated 
10 days. Where the sign plus and minus is used it indicates that one 
of the tubes showed growth while the others did not. 



TABLE V. THERMAL DEATH POINT 



(Incubated 10 days) 
!46oC 



8 B. lathyri 
43C " 
41K " 
40g, " 
22 " " " 
42 Ps. phaseoli 



Sweet Pea- 



England 
— Delaware 
Fed Clover 
Bean 
Soy Bean 
Bean 
4J- Ps. campestris Cabbage 
47 B. coli Human Intestine, Pa. 



+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 



52oC 1 54oC 



48oC[50oC 
+ 
+ 
+ 

+ 



+1 +1 +1 - 



35 

Optimum Temperature. The optimum temperature lies between 
28° to 30° centigrade. 

At 37y 2 ° C. no growth occurs on any of the nutrient broths, or 
nutrient sugar broths. Lower temperatures than 20° C. have not been 
tried. 

10. Relation to Oxygen. Bacillus lathyri n. sp. altho not strictly- 
aerobic, makes so little growth in the absence of oxygen that it prop- 
erly should be classed as an obligate aerobe. On stab cultures in dif- 
ferent media, altho the line of stab is visible, yet the growth is very 
weak. In the arm of the fermentation tube little growth was mani- 
fested, and at the end of the 5th day the line of demarcation in growth 
between that in the bowl and that in the arm was very distinct. 

11. Production of Ferments. The presence of a peptonizing 
enzyme is indicated by the liquifaction of gelatin ; the time required, 
viz. two or three months for total liquefaction would indicate that this 
proteolytic enzyme is produced very sparingly. 

Diastase is also produced which likewise is very weak in its action. 
The production of glucose cannot be demonstrated in a starch bouil- 
lon culture till the end of the second week. On potato plugs three 
months old, glucose may be shown to be present to a considerable ex- 
tent, while check plugs of the same age showed no reduction of copper 
in Fehling's solution. 

It is difficult to say whether rennet is present or not. In several 
cultures the coagulation in milk showed before there was an acidity 
strong enough (apparently) to be the cause of curdling. "Whether the 
different acids produced vary in their coagulating power has not been 
determined. 

Oxidase. If the production of gas in the presence of peroxide of 
hydrogen indicates oxidase then it is produced by each of the 32 
strains. 



Lipase is not present. 

Crystals were not found present in any of the different media 



used. 



36 

IV. Pathogenicity 
Pathogenic for Sweet pea (Lathyrus odoratus) and other Lathy- 
tus spp. for the clovers (Trifolium spp.) for soy beans (Soja spp.) 
and culii:ary beans (Phaseolus spp.) 

V. Numerical Classification 
According to the numerical system of recording the salient char- 
acters of an organism, Bacillus lathyri n. Sp. becomes B. 211. 
'2222522. 

Media Employed 

Th;> ordinary media employed, viz. the nutrient broth, nutrient 
gelatin, nutrient agar, nutrient glucose agar and other sugar agars as 
well as sugar free broth have been prepared according to the recom- 
mendation for standard methods of Water Analysis. The reaction 
expressed in Fuller's scale was +10 except in certain special media in 
which cases the formulae of the originators or certain advantageous 
modification have been followed. 

Sugar Broths were prepared by adding one percent of the dif- 
ferent sugars as well as glycerine and mannite, to sugar-free broth. 

Glycerine agar was prepared by adding four per cent of glycer- 
ine to nutrient agar. 

The following are the formulae of the different media used. 

Dunham's Solution 

Distilled water 1000.0 cc. 

Witte 's Peptone 10.0 grams 

NaCl 5.0 " 

Nitrate Broth 

Distilled water 1000.0 cc. 

Witte 's Peptone 1.02 grams 

KN0 3 (nitrite free) 2 " . 

Asparagin Solution 

Distilled water 1000.0 cc. 

Asparagin 2.0 grams 

K 2 HP0 4 1.0 " 

MgS0 4 1.0 " 



37 

Uschinsky's Solution 

Distilled water 1000.0 ce. 

Glycerine 40.0 grams 

NaCl 5.0 " 

CaCI, 0.1 " 

MgS0 4 3 " 

K 2 HP0 4 2.0 " 

Ammonium lactate 6.0 " 

Sodium asparaginate 4.0 " 

Cohn's Solution 

Distilled water 1000.0 cc. 

KH 2 P0 4 5.0 grams 

MgS0 4 6.0 " 

Ammonium tartrate 10.0 ' ' 

KC1 5 " 

Synthetic Agar, Low in Nitrogen 

Distilled water 1000.0 cc. 

Cane sugar 50.0 grams 

KH,P0 4 1.00 " 

MgSO 2 " 

Shredded agar 15.0 ' ' 

Litmus Milk. Prepared by adding to plain milk one per cent of 
a solution of azolitmin made by dissolving 1 gram of azolitmin in 40 cc. 
of distilled water and kept at 37.5° C. for 12 to 18 hours. 

The following media were used in a search for a quick differentiat- 
ing medium; silicate jelly with Fermi's solution prepared according 
to Erwin F. Smith in his work, "Bacteria in Rel. to Plant Diseases." 
Vol. pp. 37-39; starch jelly containing Uschinsky's solution as modi- 
fied by Smith. This medium gave exceptionally fine distinctions in 
coloring for the different species used as checks. B. lathyri gave a 
copious slimy yellow growth, varying in color from light straw to 
orange. The sub medium was not changed in color. Ps. phaseoli pro- 
duced a very light yellow (almost colorless) slimy and copious growth ; 
submedium slightly changed. Ps. Campestris produced a typical 
prune juice coloring similar to pneumonia sputum with an abundant 
growth ; the submedium was also browned. B. coli colored the medium 
a typical light drab. The submedium was also slightly colored. We 



3S 

consider this an excellent medium for differentiating purposes in gen- 
eral. The silicate jelly gave fairly distinctive colorings though not so 
marked as Uschinsky's medium in starch jelly. 

The use of litmus lactose in preliminary isolation work was of 
some importance in differentiating non acid producing organisms. We 
have used this medium in deep shake culture with only 1/3 the amount 
of agar viz. 5 to 6 grams, in the ascertaining of the production of acid 
and gas. We have likewise used in this work the mixture of the fol- 
lowing sugars, etc., in testing production of gas and acid in deep 
shake culture and find it of some importance, viz. 

Saccharose 2 grams 

Lactose 2 ' ' 

Dextrose 2 ' ' 

Maltose 2 " 

Mannite 2 ' ' 

Glycerine 3 cc. 

Peptone 10 grams 

Extract of beef 3 " 

Sodium Chloride 2 " 

Agar 5-6 ' ' 

Water 1000 cc. 
NaOH (and Litmus) to make neutral. 

This medium is not a delicate test for gas production, but it ia 
extremely advantageous. We see no reason why a mixture of the above 
sugars, etc. should not be used in solution in fermentation tubes for 
determining in preliminary work the production of gas. It is hardly 
probable that the presence of a certain sugar to the extent of .2% 
would inhibit the formation of gas from another sugar. So, for ra- 
pidity in determining the biochemical characters of an organism, it 
may be useful. 

SUMMARY 

1. In the foregoing work is shown the relationship existing be- 
tween the so called "streak" disease of the sweet pea (Lathyrus odor- 
atus) and a pathogenic bacterium herein described and named 
Bacillus lathyri n. sp. 

2. The disease is most active during the flowering period of the 
host, at times becoming so disastrous as to entirely destroy the crop. 
The season of heavy dew appears to be a time which favors infection.. 



39 

3. The pathogen produces somewhat similar diseases on the 
clovers (Trifolium spp.) on culinary beans (Phaseolus spp.) and on 
soy beans (Soja spp.) altho on the latter only one variety shows much 
injury among the many grown at the Experiment Station Farm, New- 
ark, Del. 

4. The disease is widespread in its distribution. In England it 
has received much comment, being popularly spoken of as the "mys- 
terious" and "dreaded streak" disease. Specimens have been re- 
ceived from England, Massachusetts, Maine, New York, and Delaware. 
The disease is reported in Ireland. 

5. Massee and Chittenden (England) have assigned Thielavia 
basicola as the causal organism. Dyke has referred it to a Macro- 
sporium. The foregoing work and work done by my associate J. J. 
Taubenhaus indicate that the two fungi above mentioned are in iu 
way related to "streak" of the sweet pea. 

6. The parasitic bacterium is a yellow rodshaped organism having 
rounded ends, and in size somewhat smaller than the average. It is 
peritrichiate in the distribution of its flagella ; ordinarily it moves rap- 
idly in liquid media. 

7. Cross inoculation on the several hosts with different sources of 
the organism, together with morphological, cultural and biochemical 
studies on 32 different sources indicate the organism to be a pathogen 
heretofore not described. 

8. Under the numerical system of noting the salient features of 
an organism Bacillus lathyri n. sp. becomes B. 211.2222522. 

ACKNOWLEDGMENTS. 
The writer is deeply indebted to Dr. John M. MacFarjane and 
Dr. John W. Harshberger for the helpful suggestions and kindly crit- 
icism offered throughout the work. To my associate Dr. J. J. Taub- 
enhaus much credit is due for the able assistance rendered, at times 
necessitated by the close relationship of our projects. Among the 
many others who have contributed by furnishing data, specimens, 
seeds and pecuniary assistance, the following deserve grateful ac- 
knowledgment: Messrs. Wm. Sim, Cliftondale, Mass; T. A. Weston, 
Orpington, England ; W. Atlee Burpee of Philadelphia, Pa. ; C. C. 
Morse & Co., San Francisco, Gal, ; A. T. Boddington, New York City, 
and Braslan Seed Growers Co., San Jose, Cal. 



40 



BIBLIOGRAPHY 



1 Burrill, T. J. Trans. 111. State Hort. Society for 1877, p. 114, and 1878 p. 80. 

2 Hartig, Eobert. Lehrbuch der Baumkrankheiten. Berlin, Julius Springer, 

1882, pp. VIII, 198, mit 186 figuren auf 11 Lithographirten Tafeln und 
86 Holzschnetten. 

3 Prillieux, Ed. Maladies des plantes agiicoles et des arbres fruitier et forest- 

iers causee par des parasites vegetaux. Paris, Librarie de Firmin. Didot 
et Cie, 56 Eue Jacob, 1895, pp. XVI, 421, Tome 1. 

4 Comes, O. Crittogramia Agraria. Vol. Unieo, Napoli, Eiccardo Marghieri di 

Gius, 77 Galleria Umberto I. 1891 pp. 600. Cap. XXX, Schizomiceti, pp. 
493-530. 

5 Sorauer, Paul. Die Schaden der einheimischen Kulturpflanzen durch tierische 

und pflanzliche Schmarotzer sowie durch andere Einflusse. Berlin, Paul 
Parey, 1888 pp. VII 250. Die Spaltpilze (Schizomycetes) pp. 152-154. 

6 Savastano, Luigi — Tubereolosi iperplasie et trmori del' olivo. Ann. E. Sc. 

Sup. Agr. Portici, 1887, Vol. V. fasc. 4. 

7 Arthur, J. C. Histology and biology of pear blight. Proc. Phil. Acad. Nat. 

Sci. 1886, pp. 322-341. 

8 Beyerinck. Die Bakterien der Papilionaceen-knollchen. Bot. Zeitung, Jahrg. 

XLVI, 1888. 

9 Wakker, J. H. Vorlaufige Mittherlungen uber Hyacinthenkrankheiten. Botan. 

Centralblatt. Nov. 1883, Bd. XIV, No. 23, pp. 315-316. 

10 Smith, Erwin P. Are there bacterial diseases of plants? A consideration of 

some statements in Dr. Alfred Fischer's Vorlesungen uber Bakterien. 
Centralbl. f. Bakt. etc. 2 te Abt., Bd. v 1899, pp. 271-278. 

11 Taubenhaus, J. J. A study of some Gloeosporiums and their relation to a 

sweet pea disease. Phytopathology, Ithaca, N. Y. Dec. 1911. 

12 Massee, George. The Sweet Pea Annual 1909, p. 222, Official Organ of the 

Nat. Sweet Pea Society, Brentford, England. 

13 Chittenden, F. J. " On Some Plant Diseases New to or Little Known in Brit- 

ain" Journ. Eoyal Hort. Society, Vol. 36, Pt. 3, pp. 545-550, 1912. 

14 Chittenden, F. J.— The Sweet Pea Annual, 1912, pp. 14-24. 



41 

15 Massee, George. "A Disease of Sweet Peas, Asters and Other Plants" (Thie- 

lavia basicola Zopf.) Eoy. Bot. Garden, Bui. of Miscl. Information No. 1, 
1912, p. 44-52. 

16 Dyke. Gard. Chron. 3 Ser. 51 (1912) Nos. 1308 p. 36; 1309 p. 52-53; 1311 pp. 

84-85. 

17 Manns, Thos. F., — "A Bacterial Disease of the Sweet Pea and Clovers." Paper 

presented before the American Phytopathological Society, Jan. 3, 1913, 
at Cleveland, Ohio. 

18 Layley, Dorothy M., — A Preliminary Note on a New Bacterial Disease of 

Pisnm sativum. Proceed, of the Eoyal Society (England) B. Vol. 86. 
Jan. 23, 1913. 

19 Manns, Thos. F. and Taubenhans, J. J. — " Streak, A Bacterial Disease of the 

Sweet Pea and Clovers. ' ' The Gardener 's Chronicle, London, Eng. Vol. 
53, pp. 215-216, 1913. 

20 Bain, S. M. and Essary, S. H. — A New Anthracnose of Alfalfa and Eed 

Clover. Jour. Myc. 12. 192-192, 1908. 

21 Selby, A. D. and Manns, T. F. — Studies in Diseases of Cereals and Grasses. 

Bui. Ohio. Agr. Exp. Sta. 203-187-236; 1909. 

22 Smith, Elizabeth H. The Blossom End Eot of Tomatoes. Mass. Agr. Exp. 

Sta. Tect. Bui. No. 3, (Apr. 1907). 

23 Earle, F. S., Black Eot or Blossom End Eot (Tomato) Ala. Exp. Sta. Bui. 108, 

1900 p. 19. 

24 Stuart, William. A Bacterial Disease of Tomatoes, Ind. Exp. Sta. Eeport 13, 

1900, p. 13. 



42 



DESCRIPTION OF PLATES 

PLATE I. Longitudinal section from stem of red clover through a beginning lesion 
infected with the "streak" organism (Bacillus lathyri. n. sp.) of sweet pea. 
The organism in the stems of clover penetrate only a few cells in depth, 
seldom apparently reaching the woody tissue. 

Drawn from a photomicrograph printed on glossy Velox, and partly filled 
in by the aid of the microscope. The background was washed out with a 
solution of potassium syanide. X330. 

PLATE II. Cross section of the stem of sweet pea through a beginning lesion 
of the ' ' streak ' ' disease caused by Bacillus lathyri. The lesion gradually 
sinks into the stem until the woody tissue is reached. The organisms do not 
apj ear to enter the vessels. The mesophyll is gradually broken down, the 
lesion extending along the entire stem, producing familiar ' ' streak ' ' effect. 

The groups of bacteria show as darkened areas in the mesoiihyll at points 
marked B. Photomicrograph X75. 

PLATE III. Photograph from a colored plate showing typical symptoms of the 
' streak'' disease in various degrees of progress on sweet pea. The disease 
is not limited to the stems, but occasionally attacks the foliage, producing 
v ater-soaked areas and blight similar to that of the bean disease. The pods 
are also attacked, causing dark purple lesions which cannot be mistaken for 
other diseases of the sweet pea. In advance stages of infection the plants 
succumb to the attack, dying prematurely. 

From a drawing by Miss Martha Chamberlin. 

PLATE IV. Showing a "streak" disease of Lima bean caused by B. lathyri. Ex- 
tensive cultures were run from lesions on stems of beans with the result that 
B. lathyri was found as the infesting organisms. The organism from this 
source when inoculated onto red clover produced infections and lesions iden- 
tical with that of the organism from clovers or sweet pea. 

Photographed natural size. 

PLATE V. In this plate are shown the typical symptoms of the bean blight disease 
caused by Ps. phaseoli as described by Dr. Erwin F. Smith. On the leaves 
are formed large water-soaked areas which, upon drying up, produce the char- 
acteristic blight. The pods likewise show large water-soaked lesions which 
sink in and turn brown or even purple when the infection is shallow. Intro- 
duced here for comparison with the "streak" disease. Slightly enlarged 
in photographing. 

PLATE VI. Showing the "streak" disease produced by B. lathyri on alsike 
clover. The plant at the left shows dark lesions throughout the stem; the 



43 

petioles become so badly diseased as to blight and dry up causing wilting of 
the leaves. The plant at the right is healthy and used here for comparison. 

Reduced to 4 /5 natural size. 

PLATE VII. Comparing "streak" infected stems of red clover (three stems) 
at the left with healthy stem at the right. The blighted leaf at the left is 
brought about by the disease attacking the petiole. 

Enlarged to 1 1-2 times nat. size. 

PLATE VIII. Showing progress of the "streak" disease on red clover. The 
stem at the left is healthy ; the stem next to the one at the left shows be- 
ginning lesions ; the three stems to the right show further progress of the 
disease. Enlarged 1 1/3 times natural size. 

PLATE IX. Showing the type of leaf infection produced by B. lathyri on Mam- 
moth clover. The three leaves to the right and below show different stages 
in the progress of the disease. The healthy leaf at the left upper corner is 
introduced for comparison. Two-thirds natural size. 

PLATE X. Showing artificial infections produced in laboratory on red clover 
by use of sources of B. lathyri respectively from Red Clover, Alsike clover, 
Sweet Pea from England and Sweet Pea from Delaware. The types of in- 
fection are similar in each case. Previous to infection the plants were 
thoroly disinfected with potassium permanganate, then after 24 hours care- 
fully washed off with sterile water, following which they were placed under 
bell jar to note whether any natural infection would arise. The plants were 
then covered by atomizer spray of 48 hour cultures. The infection followed 
in 5 to 9 days. Two-thidrs natural size. 

PLATE XL Artificial infections produced in the laboratory by use of pure cult- 
ures by B. lathyri from alsike, alfalfa and soy bean. The method of inocu- 
lation was similar to that described under Plate XL 

Two-thirds natural size. 

PLATE XII. Artificial infections produced in the laboratory by use of pure cult- 
ures of B. lathyri from white pole bean, cowpea and soil suspected of carry- 
ing the organism. Two-thirds natural size. 

PLATE XIII. Showing soy beans infected in field with the streak disease caused 
by B. lathyri. Only one variety showed much loss from this disease, in which 
case 10% of the plants were destroyed. The lesions sink deep into the stem 
near the ground and the plants blow over similar to that in potato attacked 
with black leg late in the season. 
Photograph taken at the Experiment Station Farm, Newark, Del., Oct. 1912. 

PLATE XIV. Showing "streak" lesions on pods of soy bean and also on the 
lower part of the stem in the plant at the left. 

Slightly reduced in photographing. 



44 

PLATE XV. Showing lesions of the ' ' streak ' ' disease on the stems of soy beans. 
When the lesion becomes deep the stems are so weakened that the plant blows- 
over very easily. Enlarged 1 1-2 times in photographing. 

PLATE XVI. Showing artificial infection on tomato by a yellow bacterium with 
morphological, cultural, and biochemical characteristics similar to that of 
the ' ' streak ' ' disease organism. No cross inoculation work was carried out 
from sweet pea to tomato and vice versa. In the natural infection in field 
there were associated quite frequently a Fusarium and an Alternaria. See 
Plate XVII. Photograph natural size. 

PLATE XVII. Showing appearance of fruit rot or ' ' point rot ' ' of tomato from 
natural infection in field when the surface has been sterilized and the fruit 
placed in moist chamber; quite frequently there is associated with the bac- 
terium a Fusarium and an Alternaria. The former may induce some symp- 
toms of rot; the latter is saprophytic as indicated by a series of inoculation 
work. Photographed natural size. 

PLATE XVIII. Shoeing appearance of the streak disease organism, B. laihyri. 
on nutrient agar and nutrient gelatin from Red Clover, two different sources,, 
viz. No. 15 and No. 13a. The colonies on nutrient agar have an early ten- 
dency to become stellate. 

Reduced to % natural size in photographing. 

PLATE XIX. Showing appearance of the "streak" disease organism B. laihyri 
on nutrient agar and nutrient gelatin from sweet pea, bean, and a forage 
species of Lathyrus, sources No. 5, 40 and 26f. 

The colonies on nutrient agar have an early tendency to become stellate. 
Reduced to % natural size in photographing. 

PLATE XX. Showing appearance of the "streak" disease organism B. laihyri 
on nutrient gelatin from sweet pea; sources 7A', 4', and S '. The colonies 
on nutrient agar have an early tendency to become stellate. 

Reduced to % natural size in photographing. 

PLATE XXI. Showing appearance of the "streak" disease organism B. laJiyri 
on nutrient agar from Red Clover, Soy bean and an organism with similar 
morphological, cultural and biochemical characteristics from the point rot 
of tomato. 

The surface colonies on nutrient agar all have an early tendency to be- 
come stellate. Reduced to Y-2 natural size in photographing. 




Plate I 




% 



Plate II 




Plate III 




Plate IV 




Plate V 




Plate VI 




Plate VII 




Plate VIII 











• 



•^"•^jsp 1 



Plate IX 












Plate X 










Plate XI 




ftx*(. ■■#4. x 



**«t 






Plate XII 




Plate XIII 




Plate XIV 




Plate XV 




Plate XVI 




Plate XVII 




■ 



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l\~z.-6(. ' Cove 





/J/9— ?1<s-t~ a^a-~~ 




Plate XVIII 




/a -. -2. -/A 




- - - 




/ o - ^ - ^, 







Plate XIX 







Plate XX 




/a _ a - /<f y 




llf J ' _ "?t»*/ ^ 








Plate XXI 



